Preparation of lipase with improved ester synthesis activity by using surfactants

ABSTRACT

The present disclosure discloses a method for preparing lipase with high ester synthesis activity by using surfactant, belonging to the field of enzyme engineering. The present disclosure provides a method for obtaining a lipase with high ester synthesis activity by adding different surfactants with different concentrations in a lipase aqueous solution and then freeze-drying. The lipase meets the requirement in non-aqueous catalysis. Mixing a variety of lipase with no ester synthesis activity or low activity at suitable concentration and an appropriate concentration of the surfactant in the solution can produces lipases with significantly improved ester synthesis activity, meanwhile changing the hydrolytic activity of the lipase. Increased ester synthesis activity makes lipase more suitable for industrial applications.

TECHNICAL FIELD

The disclosure herein relates to the preparation of lipase with improvedester synthesis activity by using surfactants, which belongs to thefield of enzyme engineering.

BACKGROUND

Lipase (triacylglycerol ester hydrolases, EC 3.1.1.3), firstly reportedin 1901, is a class of biocatalysts hydrolyzing long-chaintriacylglycerol ester at the water-oil interface and widely exists inanimals, plants and microorganisms. Besides the hydrolysis of esterbonds, lipases can catalyze esterification and transesterificationreaction in non aqueous environments and therefore gain great interestin the field of biological chemical industry. As being able tofacilitate very special chemical transformation, lipases are gainingmore and more interest in several industries such as food, detergents,cosmetics, organic synthesis, pharmaceutical. For example, they can beused in progressing food, wastewater treatment, synthesis of biologicalsurfactants, treatment of wood cellulose pulp paper resin andbiosynthesis of chiral drugs. Lipases also have advantages of goodstability and high conversion efficiency and they have application valuein the production of aromatic esters, biodiesel and chiral compounds.

Most lipases can catalyze hydrolysis of ester bonds. However, only fewcatalyzes ester synthesis reaction in non-aqueous media and mostmicrobial lipases do not display apparent activity when catalyzing estersynthesis reaction in non-aqueous media. Several heterologous expressedlipases display only remarkable hydrolysis activity but little abilityto catalyze synthesis of esters in comparison with wild-type lipases.The industrial application of lipases is hampered by their pooractivities catalyzing ester synthesis reaction.

Several protein-modified methods, such as enzyme immobilization andpretreatment with organic solvent, have been adapted to adjust orimprove activity of enzyme. But these methods cannot change catalyticperformance of lipases essentially. Besides, surfactants can adjustcatalytic performance and generally improve the lipase activity ofhydrolysis. Employing surfactants to enhance lipase activity innon-aqueous media have also been reported decades ago, includingformation of surfactant-coated enzyme or reverse micelle system, both ofwhich will improve the solubility of enzymes in non-aqueous media, andmolecular bio-imprinting technologies—lyophilize lipases in the presenceof surfactants and wash them off afterwards. Although sometimeseffective, these methods are complicated and time-consuming in practiceand cannot be applicable to all the lipases. Developing astraightforward, versatile method to improve the activity of lipasecatalyzing ester synthesis in non-aqueous has a significant practicalvalue.

SUMMARY

To solve the problems above, the present disclosure provides a methodfor preparation of the lipases meeting the requirement of practicalapplication in non-aqueous catalysis, which have high ester synthesisactivity, by changing microstructures of lipases via the interactionbetween surfactants and soluble lipases in aqueous environments andlyophilizing mixture afterwards. The present disclosure also provides amethod which can be applicable to other lipases.

The present disclosure provides a method for enhancing ester synthesisactivity of lipases by using surfactants. The present disclosure relatesto modify lipases by adding surfactants and obtain lipases bylyophilization, which are able to catalyze synthesis of esters.

The present disclosure solves the problem that many recombinant lipasesdo not display ester synthesis activity, and lays the foundation forexpanding the industrial application of lipase. The present disclosureis obviously different from the existing methods for modifying lipase byusing surfactants to obtain high ester synthesis activity, and has goodoperability and wide applicability.

The method for enhancing ester synthesis activity of lipases is todirectly add surfactants to the lipase solution and mix afterwards,which can provide a hydrophobic environment enabling soluble lipases toregain the ester synthesis activity by changing the microstructure oflipases.

In an embodiment of the present disclosure, the ester synthesis activitydescribed refers to the activity of catalyzing synthesis of esters in anon-aqueous phase.

In an embodiment of the present disclosure, the aqueous solution of thelipase described refers to the solution obtained by dissolving thelipase (powder) directly in an aqueous solution.

In an embodiment of the present disclosure, the aqueous solutiondescribed can be water and other buffer solutions.

In an embodiment of the present disclosure, the buffer solution can bephosphate buffer, citrate buffer, acetate buffer, Tris buffer and thelike.

In an embodiment of the present disclosure, the concentration of buffersolution ranges from 25 to 100 mmol·L⁻¹ pH 6.5-8.0.

In an embodiment of the present disclosure, the buffer is a 25 mmol·L⁻¹phosphate buffer pH 7.5.

In an embodiment of the disclosure, the lipases described include, butare not limited to, Rhizopus chinensis lipase r27RCL, Rhizoupus oryzaelipase ROL, Pseudomonas cepacia lipase PCL, Candida antarctica lipaseCALB, and the like.

In an embodiment of the disclosure, the lipase can be a lipase that isheterologously expressed, a lipase expressed by a recombinantmicroorganism, or any other lipases that have lower or no estersynthesis activity relative to the wild-type lipase.

In an embodiment of the present disclosure, the protein concentration ofthe pure enzyme in the soluble lipase solution described ranges from 0.1and 0.4 mg·mL⁻¹ to ensure that lipases interact with surfactantscompletely.

In an embodiment of the present disclosure, the surfactant described canbe one or a combination of a zwitterionic surfactant, a nonionicsurfactant, a cationic surfactant, and the like. Different surfactantshave different effects on different lipases. But surfactants describedare not limited to the above-mentioned surfactants.

In an embodiment of the present disclosure, the zwitterionic surfactantsare 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),polyethylene glycol octylphenyl Ether (Triton X-100), 1-myristyl2-acyl-cis-propyltri-phosphatidylcholine (LPC14), 1,2-dihexanoyllecithin (DiC6PC), 1-(LPC16),1-Lauramate-2-acyl-cis-propyltri-phosphatidylcholine (LPC16),1-myristyl-2-acyl-cis (LPG12),1-caramom-2-acyl-cis-propyltriyl-phosphoric acid-1-glycerol (LPG14),1-palm-2-acyl-cis (LPG16), 1,2-diheptanoyl-S-glycero-3-phosphorylcholine(DHPC), or a combination of two or more.

In an embodiment of the present disclosure, the nonionic surfactantsdescribed are n-dodecyl-ß-D-maltopyranoside (DDM), octaethylene glycolmonododecyl ether (C12E8), n-decyl-ß-D-maltopyranoside (DM),n-undecyl-ß-D-maltopyranoside (UDM), n-octyl-ß-D-glucopyranoside (OG),octaethylene glycol monodecyl ether (C10E8), or a combination of two ormore.

In an embodiment of the present disclosure, the cationic surfactantsdescribed are hexadecyl trimethyl ammonium bromide (CTAB) and the like.

In an embodiment of the present disclosure, the surfactant aqueoussolution can be stored at 4° C. for a short time.

In an embodiment of the present disclosure, the protein solution and thesurfactant solution are mixed at a volume ratio ranging from 1:0.5 to1:2.

In an embodiment of the present disclosure, the protein solution and thesurfactant solution are mixed at a volume ratio of 1:1.

In an embodiment of the present disclosure, the final concentration ofthe surfactant ranges from 0.1 times the critical micelle concentration(0.1×CMC) to 200 times the critical micelle concentration (200×CMC).

In an embodiment of the present disclosure, the final concentration ofthe surfactant ranges from 10 times the critical micelle concentration(10×CMC) and 200 times the critical micelle concentration (200×CMC).

In an embodiment of the present disclosure, the final concentration ofthe surfactant is 100 times the critical micelle concentration(100×CMC).

In an embodiment of the present disclosure, the amount of surfactantadded is high enough to make the final concentration range from 1 to 500mM.

In an embodiment of the present disclosure, the amount of surfactantadded is high enough to make the final concentration reach 10 mM.

In an embodiment of the present disclosure, the addition of surfactantscan further enhance ester synthesis activity and/or total recovery ofester synthesis activity of Rhizopus chinensis lipase r27RCL, Rhizoupusoryzae lipase ROL, Pseudomonas cepacia lipase PCL, Candida antarcticalipase CALB and the like.

In an embodiment of the present disclosure, the method comprises thestep of removing the water after mixing.

The second object of the present disclosure is to provide a lipasepreparation with improved activity catalyzing ester synthesis in anon-aqueous phase, wherein the lipase preparation is prepared bydissolving a lipase in an aqueous solution, directly adding asurfactant, mixing, and then removing the water.

In an embodiment of the present disclosure, the step of removing thewater described can be the drying, and lyophilizaton.

In an embodiment of the present disclosure, the aqueous solutiondescribed can be water and other buffer solutions.

In an embodiment of the disclosure, the lipases described can be alipase that is heterologously expressed, a lipase expressed by arecombinant microorganism, or any other lipases that have lower or noester synthesis activity relative to the wild-type lipase.

The third object of the present disclosure is to provide the applicationof the lipase described in ester synthesis reaction.

In an embodiment of the present disclosure, the ester synthesis reactionis a ester synthesis reaction in non-aqueous phase.

The fourth object of the present disclosure is to provide theapplication of lipases described in the field of food, chemical,biological, pharmaceutical, environmental, and petroleum.

In an embodiment of the present disclosure, the application includes,but is not limited to, detergents production, cosmetics production,wastewater treatment, bio-surfactants production, wood cellulose pulppaper resins treatment, biosynthesis of chiral drugs, aromatic esterproduction, biodiesel production, chiral compound production and thelike.

Beneficial Effect of the Present Disclosure

(1) Conformation and function of protein are closely related to theenvironments around. The present disclosure provides a method ofproducing lipases with high ester synthesis activity by in vitroadjustment of lipase activity by simulating cellular membranehydrophobic environment using surfactants.

(2) The method of the present disclosure is applicable to Rhizopuschinensis lipase r27RCL expressed by Pichia pastoris, commercialRhizoupus oryzae lipase ROL, commercial Pseudomonas cepacia lipase PCL,commercial Candida antarctica lipase CALB and the like. The methoddescribed can help lipase regain ester synthesis activity or increaseester synthesis activity by 8-30 times, and enable the total recovery oflipase ester synthesis activity reach 105%-1733%.

DETAILED DESCRIPTION

Determination of Lipase Activity

(1) Determination of Hydrolytic Activity of the Lipase Usingp-Nitrophenyl Palmitate (pNPP) as Substrate

Hydrolysis of pNPP catalyzed by lipase generates p-nitrophenol andpalmitic acid. p-nitrophenol appears yellow in the buffer at pH 8.0 withthe maximum absorption peak at 410 nm. Determination of hydrolyticactivity can be achieved by measuring the absorbance at 410 nm.

The substrate solution A: 50 mmol·L⁻¹ sodium phosphate buffer (pH 8.0)containing 1.16 g·L⁻¹ sodium deoxycholate and 0.56 g·L⁻¹ arabic gum.Substrate solution B: 0.015 g pNPP is dissolved in 5 mL isopropylalcohol. Substrate solution A and substrate solution B are mixed thenstored for use afterwards.

Termination solution: 40 g·L⁻¹ NaOH, 93.05 g·L⁻¹ EDTA sodium. Add 62 uLtermination solution to the reaction mixture when stopping the reaction.

Determination Method:

Add 0.1 mL appropriately diluted enzyme solution, which is substitutedby inactivated enzyme solution as the control, to 2.4 mL substratesolution above, incubate the mixture at 40° C. for 2 min and measure theabsorbance at 410 nm.

Definition of Enzyme Activity:

one hydrolytic activity unit is defined as the amount of enzyme thatcatalyzes the formation of 1 μmol of p-nitrophenol in 1 minute at 40° C.

enzyme activity (U·mL⁻¹)=(V×A ₄₁₀×10⁶)/(ε×t×V′)  Calculation equation:

where V is the volume of the reaction mixture (mL), E is the molarextinction coefficient (mL·mmol⁻¹), t is the reaction time (min) and V′is the volume of the enzyme solution (mL).

(2) Determination of Ester Synthesis Activity of the Lipase by GCAnalysis

Reaction Substrates:

Substrate solution A: 48.5 mL of n-octanoic acid is dissolved in 250 mLof n-heptane in a volumetric flask.

Substrate solution B: 17.5 mL of absolute ethanol is dissolved in 250 mLof n-heptane in a volumetric flask.

Internal standard: 2-hexanol/n-heptane (35 g·L⁻¹).

Standard sample: 10 g of n-octanoate is dissolved in 1000 mL ofn-heptane in a volumetric flask.

Method of Determination

1 mL substrate solution A and substrate solution B are added in a 5 mLEppendorf tube respectively, then 20 mg lipase powder (or lyophilizedpowder) is added. The reaction is carried out at 40° C. and with shakingat 150 rpm for 30 min. Remove the lipase powder by centrifugation ormembrane filtration. Then 0.1 mL of internal standard is added in 0.4 mLof filtrate or supernatant and mixed. Measure the content of n-octanoatein the mixture above by GC analysis.

The gas chromatograph (6820, Agilent Instruments) is equipped with aAC20 (PEG20000) capillary column and a FID detector. Nitrogen was usedas the carrier gas. The oven temperature was programmed to start at 90°C. for 1 min and then be elevated to 200° C. for 5 min at 10° C.·min⁻¹.The injector and detector temperatures were set at 250° C.

Definition of enzyme activity: One unit of ester synthesis activity isdefined as the amount of enzyme that esterifies 1 micromole ofn-octanoate per min.

Calculation Equation:

${{enzyme}\mspace{14mu} {activity}\mspace{14mu} \left( {U \cdot {mg}^{- 1}} \right)} = {\frac{A_{sam}}{A_{sta}} \times S_{sta} \times V \times 10^{6} \times \frac{1}{172} \times \frac{1}{30} \times \frac{1}{m}}$

A_(sam)—the ratio of the peak areas of the sample for testing to theinternal standard;

A_(sta)—the ratio of the peak areas of the standard sample to theinternal standard;

S_(sta)—the concentration of the standard sample (g·L⁻¹);

V—the volume of the reaction mixture (L);

m—the amount of lipase in the reaction mixture (mg).

Specific activity: the activity of lipase per milligram of total proteinin the lipase preparation.

Total recovery: the percentage of the activity after a treatment to thatbefore the treatment

Example 1: Method of Operation for Enhancing Lipase Ester SynthesisActivity

Add an equal volume of high-concentration surfactant solution into theenzyme solution. The final protein concentration was 0.25 mg·mL⁻¹ andthe surfactant concentration was 10 mM. After mixed thoroughly, themixture was lyophilized and kept in dry condition.

Example 2: Regain of the Ester Synthesis Activity of Commercial r27RCL

Commercial lipase r27RCL was purchased from Jiangsu Yiming BiologicalTechnology Co., Ltd, which has high hydrolytic activity and low estersynthesis activity. Dissolve the r27RCL powder in water and centrifugeto obtain the supernatant containing the r27RCL lipase to adjust theester synthesis activity (the final protein concentration was 0.2mg·mL⁻¹ and the final concentration of surfactant was 10 mM, which isbetween 0.1×CMC^(˜)200×CMC of the different surfactants in Table 1). Asshown in Table 1, addition of LPC14 increased the ester synthesisspecific activity from 5.5 U·mg⁻¹ to 47.6 U·mg⁻¹ and the total recoveryof ester synthesis activity was 769% while the hydrolytic specificactivity did not show significant change.

TABLE 1 Ester synthesis activity and hydrolytic activity of r27RCL.Specific activity Total recovery surfactant (U · mg⁻¹) (%) Ester blank5.5 100 synthesis CTAB 0 0 activity DiC6PC 15.9 256 C12E8 22.2 372 LPC1447.6 769 DDM 31.4 423 CHAPS 13.0 105 Triton X-100 12.9 192 Hydrolyticblank 115.7 100 activity CTAB 0 0 DiC6PC 104.2 87 C12E8 117.1 101 LPC14116.1 97 DDM 135.0 94 CHAPS 132.6 109 Triton X-100 116.6 93

Example 3: Regain of the Ester Synthesis Activity of Commercial ROL

Commercial lipase ROL was purchased from Sigma-Aldrich Co., Ltd. ROL isa non-immobilized lipase. Dissolve the ROL powder in water andcentrifuge to obtain the supernatant containing the ROL lipase thatwould be regulated to improve its ester synthesis activity by adjustingthe final protein concentration to 0.2 mg·mL⁻¹ and adding surfactants tothe final concentration of 10 mM. As shown in Table 2, addition of LPC14increased its ester synthesis specific activity from 0.4 U·mg⁻¹ to 12.2U·mg⁻¹ and the total recovery of ester synthesis activity was 1733%.

TABLE 2 Ester synthesis activity and hydrolytic activity of ROL.Specific activity Total surfactant (U · mg⁻¹) recovery (%) Eester blank0.4 100 synthesis CTAB 0 0 activity DiC6PC 3.11 733 C12E8 2.6 400 LPC1412.2 1733 DDM 2.8 333 CHAPS 2.6 400 Triton X-100 1.7 200 Hydrolyticblank 18.5 100 activity CTAB 0 0 DiC6PC 15.2 76 C12E8 23.7 82 LPC14 33.1106 DDM 42.7 116 CHAPS 23.6 81 Triton X-100 34.9 99

Example 4: Regain of the Ester Synthesis Activity of Commercial PCL

Commercial lipase PCL was purchased from Sigma-Aldrich Co., Ltd. PCL isa non-immobilized lipase. Dissolve the PCL powder in water andcentrifuge to obtain the supernatant containing the PCL lipase thatwould be regulated to improve the ester synthesis activity by adjustingthe final protein concentration to 0.2 mg·mL⁻¹ and adding surfactants tothe final concentration of 10 mM. As shown in Table 3, addition of DDMincreased its ester synthesis specific activity from 2.5 U·mg⁻¹ to 55.6U·mg⁻¹ and the total recovery of ester synthesis activity was 1435%.

TABLE 3 Ester synthesis activity and hydrolytic activity of PCL.Specific activity Total surfactant (U · mg⁻¹) recovery (%) Ester Blank2.5 100 synthesis CTAB 0 0 activity DiC6PC 7.1 260 C12E8 4.6 130 LPC1444.6 1260 DDM 55.6 1435 CHAPS 4.7 130 Triton X-100 6.3 174 Hydrolyticblank 214.8 100 activity CTAB 75.5 29 DiC6PC 308.2 140 C12E8 281.1 98LPC14 321.8 119 DDM 327.7 111 CHAPS 248.0 101 Triton X-100 364.5 131

Example 5: Increase of the Ester Synthesis Activity of Commercial CALB

Commercial lipase CALB was purchased from Sigma-Aldrich Co., Ltd. CALBis a non-immobilized lipase. Dissolve the CALB powder in water andcentrifuge to obtain the supernatant containing the CALB lipase thatwould be regulated to improve the ester synthesis activity by adjustingthe final protein concentration to 0.12 mg·mL⁻¹ and adding surfactantsto the final concentration of 10 mM. As shown in Table 4, addition ofDDM increased the ester synthesis specific activity from 7.8 U·mg⁻¹ to101.9 U·mg⁻¹ and the total recovery of ester synthesis activity was700%.

TABLE 4 Ester synthesis activity and hydrolytic activity of CALB.Specific activity Total surfactant (U · mg⁻¹) recovery (%) Ester Blank7.8 100 synthesis CTAB 36.6 300 activity DiC6PC 31.9 400 C12E8 62.8 467LPC14 36.8 411 DDM 101.9 700 CHAPS 5.9 44 Triton X-100 77.4 544Hydrolytic blank 1.1 100 activity CTAB 0 0 DiC6PC 0.98 85 C12E8 0 0LPC14 0.54 38 DDM 0 0 CHAPS 1.6 92 Triton X-100 0 0

Example 6: Effect of Concentration of Surfactant LPC14 on EsterSynthesis Activity of Commercial r27RCL

Commercial lipase r27RCL was purchased from Jiangsu Yiming BiologicalTechnology Co., Ltd. r27RCL is a lipase with high hydrolytic activitybut low ester synthesis activity. Dissolve the r27RCL powder in water,centrifuge to obtain the supernatant and treat the supernatant withdifferent concentrations of surfactant LPC14 (the final proteinconcentration was 0.2 mg·mL⁻¹). As shown in Table 5, ester synthesisactivity of r27RCL obtained more improvement at higher concentration ofsurfactant LPC14 (100×CMC).

TABLE 5 Effect of concentration of LPC14 on ester synthesis activity andhydrolytic activity of r27RCL. concentration Specific activity Total (xCMC) (U · mg⁻¹) recovery (%) Ester synthesis 0 5.5 100 activity 0.1 1.832 1 1.4 25 100 17.1 292 Hydrolytic 0 115.7 100 activity 0.1 120.2 101 1122.2 102 10 128.0 107 100 125.7 102

Example 7: Effect of Concentration of Surfactant LPC14 on EsterSynthesis Activity of Commercial ROL

Commercial lipase ROL was purchased from Sigma-Aldrich Co., Ltd. and isa non-immobilized lipase. Dissolve the ROL powder in water, centrifugeto obtain the supernatant and treat the supernatant with differentconcentrations of surfactant LPC14 (the final protein concentration was0.2 mg·mL⁻¹). As shown in Table 6, ester synthesis activity obtainedmore improvement at higher concentration of surfactant LPC14(10×-100×CMC).

TABLE 6 Effect of concentration of LPC14 on ester synthesis activity andhydrolytic activity of ROL. concentration Specific activity Total (xCMC) (U · mg⁻¹) recovery (%) Ester synthesis 0 0.4 100 activity 0.1 0.497 1 0.2 46 10 1.0 240 100 6.2 1517 Hydrolytic 0 18.5 100 activity 0.128.7 150 1 27.7 139 10 31.2 162 100 39.0 206

Example 8: Effect of Concentration of Surfactant DDM on Ester SynthesisActivity of Commercial PCL

Commercial lipase PCL was purchased from Sigma-Aldrich Co., Ltd. and isa non-immobilized lipase. Dissolve the PCL powder in water, centrifugeto obtain the supernatant and treat the supernatant with differentconcentrations of surfactant DDM (the final protein concentration was0.2 mg·mL⁻¹). As shown in Table 7, ester synthesis activity obtainedmore improvement at higher concentration of surfactant DDM(10×-100×CMC).

TABLE 7 Effect of concentration of DDM on ester synthesis activity andhydrolytic activity of PCL concentration Specific activity Total (x CMC)(U · mg⁻¹) recovery (%) Ester synthesis 0 2.5 100 activity 0.1 3.2 58 12.9 57 10 20.0 260 100 101.6 2776 Hydrolytic 0 214.8 100 activity 0.1499.5 106 1 434.7 99 10 756.7 114 100 773.5 246

Example 9: Effect of Concentration of Surfactant DDM on Ester SynthesisActivity of Commercial CALB

Commercial lipase CALB was purchased from Sigma-Aldrich Co., Ltd. and isa non-immobilized lipase. Dissolve the CALB powder in water, centrifugeto obtain the supernatant and treat the supernatant with differentconcentrations of surfactant DDM (the final protein concentration was0.12 mg·mL⁻¹). As shown in Table 8, ester synthesis activity obtainedmore improvement at higher concentration of surfactant DDM (100×CMC).

TABLE 8 Effect of concentration of DDM on ester synthesis activity andhydrolytic activityof CALB. concentration Specific activity Total (xCMC) (U · mg⁻¹) recovery (%) Ester synthesis 0 7.8 100 activity 0.1 4.012 1 14.3 50 10 37.3 73 100 32.6 189 Hydrolytic 0 1.1 100 activity 0.12.1 45 1 3.0 75 10 0.7 10 100 0 0

In summary, the present disclosure obtains the lipase preparation withhigh ester synthesis activity by in vitro regulating the activity of thelipase by simulating the hydrophobic microenvironment around the proteinusing surfactants. The ester synthesis activity of the lipase r27RCLexpressed by Pichia pastoris was increased from 5.5 U·mg⁻¹ to 47.6U·mg⁻¹ and the total ester synthesis activity recovery was 769% afterthe surfactant treatment. The ester synthesis activity of commercial ROLwas increased from 0.4 U·mg⁻¹ to 12.2 U·mg⁻¹, and the total activityrecovery was 1733% after the surfactant treatment. The ester synthesisactivity of commercial PCL was increased from 2.5 U·mg⁻¹ to 55.6 U·mg¹,and the total activity recovery was 1435% after the surfactanttreatment. The ester synthesis activity of commercial CALB was increasedfrom 7.8 U·mg⁻¹ to 101.9 U·mg¹, and the total activity recovery was 700%after the surfactant treatment. The concentrations of surfactant usedsignificantly affected the regain of lipase ester synthesis activity.The higher concentration (10×-100×CMC) of the surfactant can result inbetter lipase ester synthesis activity. The increase of both thespecific activity and total activity recovery indicated that theactivity regulation by simulating the hydrophobic environment of cellmembrane in vitro had a positive effect on the ester synthesis activityof lipase. This method was not only applicable to r27RCL, but also toother lipases.

The disclosure described and claimed herein is not to be limited inscope by the specific aspects herein disclosed. Any person skilled inthe art can make modifications without departing from the spirit andscope of the disclosure. The scope of protection of the presentdisclosure should therefore be defined by the claims.

What is claimed is:
 1. A method for increasing ester synthesis activityof lipase, comprising adding surfactants directly to aqueous solution ofsoluble lipase and mixing afterwards.
 2. The method for increasing theester synthesis activity of lipase according to claim 1, wherein theaqueous solution of the lipase is obtained by dissolving a lipase powderdirectly in the aqueous solution.
 3. The method for increasing the estersynthesis activity of lipase according to claim 1, wherein the aqueoussolution is phosphate buffer saline or Tris-HCl buffer.
 4. The methodfor increasing the ester synthesis activity of lipase according to claim1, wherein the surfactant is one or a combination of zwitterionicsurfactants, nonionic surfactants and cationic surfactants.
 5. Themethod for increasing the ester synthesis activity of lipase accordingto claim 1, wherein the soluble lipase comprises Rhizopus chinesislipase r27RCL, Rhizopus oryzae lipase ROL, Pseudomonas cepacia lipasePCL, or Candida antarctica lipase CALB.
 6. The method for increasing theester synthesis activity of lipase according to claim 1, wherein thesoluble lipase is a lipase that is heterologously expressed, a lipaseexpressed by a recombinant microorganism, or any other lipases that havelower or no ester synthesis activity relative to a wild-type lipase. 7.The method for increasing the ester synthesis activity of lipaseaccording to claim 1, wherein protein concentration of pure lipase inthe aqueous solution of the soluble lipase is between 0.1 and 0.4mg·mL⁻¹.
 8. The method for increasing the ester synthesis activity oflipase according to claim 4, wherein the zwitterionic surfactantscomprises 3-[3-(cholamidopropyl) dimethylamino] propanesulfonic acidinternal salt, polyethylene glycol octyl Phenyl ether, 1-myristyl2-acyl-cis-propyltriyl-phosphatidylcholine, 1,2-dihexanoyl lecithin,1-myristyl-2-acyl-cis-propyltri-2-acyl-cis-propyltriate-phosphatidylcholine,1-myristyl-2-acyl-cis-propyltriyl-phosphonic acid-1-glycerol, 1-myrist2-acyl-cis-propyltri-phospho-1-glycerol or 1,2-diheptanoyl-S-glyceryl,Glycerol-3-phosphorylcholine; the nonionic surfactant isdodecyl-ß-D-maltoside, octylglycol monobutyl dodecyl ester,decyl-ß-D-maltose Glycoside, undecyl-ß-D-maltoside,octyl-ß-D-glucopyranoside or polyoxypropylene glycol monethylether. 9.The method for increasing the ester synthesis activity of lipaseaccording to claim 4, wherein the nonionic surfactants comprisesn-dodecyl-ß-D-maltopyranoside, octaethylene glycol monododecyl ether,n-decyl-ß-D-maltopyranoside, n-undecyl-ß-D-maltopyranoside,n-octyl-ß-D-glucopyranoside or octaethylene glycol monodecyl ether. 10.The method for increasing the ester synthesis activity of lipaseaccording to claim 1, wherein final concentration of surfactant isranging from 0.1 times to 200 times critical micelle concentration andabove.
 11. The method for increasing the ester synthesis activity oflipase according to claim 1, wherein the addition of the surfactant is1-500 mM or higher at final concentration.
 12. A lipase preparation withimproved ester synthesis activity in non-aqueous phase, comprising alipase preparation obtained by dissolving the lipase in an aqueoussolution and then adding surfactant directly to the solution followed byremoving water.
 13. A method comprising adding the lipase preparationaccording to claim 12 as a catalyst in catalyzing ester synthesisreaction in non-aqueous phase; or adding the lipase preparationaccording to claim 12 as a catalyst in catalyzing a reaction in field offoodstuffs, chemistry, biology, preparation of medicine, or environmentor petroleum field.